Engineered Control Over Paracrine Interactions Between Hypoxic and Normoxic Skeletal Muscle

Introduction:: Various pathological conditions, such as peripheral artery disease and critical limb ischemia, can locally disrupt blood flow to skeletal muscle, leading to variations in oxygen supply to different regions of the tissue. The localized nature of arterial narrowing or blockage creates oxygen gradients within the tissue, resulting in sites of hypoxic muscle tissue surrounded by normoxic muscle tissue. The presence of hypoxic muscle tissue neighboring normoxic muscle tissue, and vice versa, can have important implications for the overall function of the affected muscle, especially because changes in function can be mediated by the exchange of secreted factors. However, the effect of hypoxic muscle tissue on normoxic muscle tissue and vice versa is still unknown, largely due to technical barriers in the ability to generate stable oxygen gradients to study such intercellular communication. The objective of this study was to engineer a microphysiological system (MPS) to determine the extent to which paracrine interactions between differentially oxygenated muscle tissue affect skeletal muscle phenotypes.


Materials and Methods:: Digital light processing 3D-printed molds were used to fabricate polydimethylsiloxane (PDMS) microfluidic network layers with channels for gas perfusion and reservoirs with walls for controlling paracrine interactions. Thin, gas permeable membranes were fabricated by spin coating PDMS on silicon wafers. Components were cured, cut to size, biopsy punched with ports for gas flow, and plasma bonded. A dual condition oxygen landscape was generated via continuous perfusion of compressed gases through the underlying microfluidic network channels and diffusion across the thin, gas permeable PDMS membrane on which engineered skeletal muscle tissue was cultured. Micromolded gelatin hydrogels were cured on top of the PDMS membrane to enable stable culture of aligned C2C12 myotubes in the MPSs. A 3D-printed oxygen control insert with internal gas channels and integrated barbed fittings was designed to interface with 2 wells of a standard 6-well plate (each containing one MPS) to supply equal flow of humidified gases to the channels underlying the PDMS membrane. O-rings on the well plate insert minimized media evaporation. After 9 days of myotube differentiation, ports on the MPSs were attached to the barbed fittings on the oxygen control insert and positioned in a well plate for 24 hour oxygen modulation experiments. C2C12 myotubes were lysed to determine gene expression by qPCR or fixed and stained for image analysis. Cell culture media was collected for analysis of secreted factors.


Results, Conclusions, and Discussions:: Dual condition oxygen landscapes were generated by continuously perfusing normoxic (10% oxygen) and hypoxic (0% oxygen) gas through the microfluidic channels. The gases diffused across the thin, gas permeable PDMS membrane and gelatin hydrogel to locally control the oxygen environment of the myotubes (Fig 1A-B). High wall MPSs (Fig. 1A) prevented paracrine-mediated intercellular communication between normoxic and hypoxic engineered muscle tissue while low wall MPSs (Fig. 1B) permitted exchange of paracrine factors. During 24 hour oxygen modulation experiments, one high wall and one low wall MPS were attached to the 3D-printed oxygen control well plate insert (Fig. 1C-D). Validation of the oxygen landscape was performed using platinum(II) octaethylporphyrin-ketone (PtOEPK) oxygen sensors (Fig. 1E). Hypoxic-normoxic intercellular communication in the low wall MPS uniquely increased expression of the gene for the myokine amphiregulin (AREG) (Fig.1F), a ligand for the epidermal growth factor receptor (EGFR) that has been shown to play a role in skeletal muscle wound repair and regeneration. Establishing hypoxic-normoxic paracrine interactions in skeletal muscle may provide insight into mechanisms of injury and inflammation and enable identification of novel therapeutic targets to minimize hypoxic injury and enhance muscle function or recovery.


Acknowledgements (Optional): :

References (Optional): :